Abstract

We have identified 2-methyl-6-(2-phenylethenyl)pyridine (SIB-1893) and 2-methyl-6-phenylethynyl pyridine hydrochloride (MPEP) as positive allosteric modulators for the hmGluR4. SIB-1893 and MPEP enhanced the potency and efficacy of L-2-amino-4-phophonobutyrate (L-AP4) in guanosine 5'-O-(3-[(35)S]thiotriphosphate ([(35)S]GTPgammaS) binding and efficacy in cAMP studies. These effects were fully blocked by the mGluR4 competitive antagonist (RS)-alpha-cyclopropyl-4-phosphonophenylglycine (CPPG), indicating a dependency on receptor activation. Although SIB-1893 and MPEP had no effects alone in GTPgammaS binding, effects were observed in the cell-based cAMP assay due to media-derived activation as indicated by CPPG inhibition. Positive modulation of the mGluR4 was a receptor-specific effect since SIB-1893 and MPEP had neither effects on mGluR2-expressing cells nor on the parent BHK cell line. In [(3)H]L-AP4 binding, a two-fold decrease in K(D) but not in B(max) was observed with 100 micro M SIB-1893, whereas MPEP affected neither parameter. Finally, SIB-1893 and MPEP failed to displace [(3)H]L-AP4 binding. Taken together, these data identify positive allosteric modulators for the hmGluR4.

Effect of SIB-1893 on L-AP4-induced [35S]GTPγS binding in hmGluR4 membranes. Data are given as CPM±s.e.m. from a representative experiment performed in triplicate. Nonspecific binding (∼3600 CPM) has been subtracted.

Potentiating effect of SIB-1893 in [35S]GTPγS binding on hmGluR4 membranes. The L-AP4 response enhanced by SIB-1893 at 10–100 μM was completely blocked by 1000 μM CPPG. SIB-1893 alone failed to increase binding above basal level. The data are mean±s.e.m from three to six independent experiments each performed in quadruplicate. Significant differences from basal: *P<0.05 and from SIB-1893 enhanced L-AP4 response; #P<0.05 (ANOVA followed by Dunnett's test).

Inhibition of forskolin-stimulated cAMP formation. In hmGluR4-expressing BHK cells, CPPG blocked the concentration-dependent effects observed with SIB-1893 alone and together with 0.1 μML-AP4. In rmGluR2-expressing BHK cells, the L-glutamate response was not augmented by SIB-1893 and no effects of SIB-1893 alone were observed. In the parent BHK cell line, L-AP4 and SIB-1893 produced no effects. The data are mean±s.e.m. of three independent experiments performed in triplicate and are expressed as % inhibition of 10 μM forskolin-stimulated cAMP production. Significant differences: *P<0.05 (ANOVA followed by Dunnett's test).